The ultimate goal of the proposal presented herein is to use E. coli as whole cell biocatalysts for the production of a wide variety of TDP-deoxysugars including di- and tri-deoxysugars, amino sugars and branched-chain sugars. These specialized activated hexoses are found as important structural components throughout plant and microbial secondary metabolites often playing a crucial role in conferring activity in bioactive natural products such as antibiotics and anticancer therapeutics. We propose to investigate a novel approach to produce rare TDP-deoxysugars in Escherichia coli through metabolic engineering. By combining genetic mutations which separately lead to increased bioavailability of glucose-6-phosphate (G6P; an intermediate of TKDG) and TDP-4-keto-6-deoxy-D-glucose (TKDG; an intermediate of TDP-deoxysugars) we plan to increase the accumulation of the TKDG precursor beyond previous reports. Additional over-expression of endogenous TKDG biosynthetic proteins may further optimize TKDG production. Finally, exogenous TDP-deoxysugar biosynthetic genes will be introduced into the strain to convert the accumulated TKDG pool into specific TDP-deoxysugars. Specifically, in Phase I we will demonstrate the feasibility of TDP-deoxysugar production in E. coli through by 1) developing analytical methods for the isolation, purification and characterization of TDP-deoxysugars produced by E. coli, 2) metabolically engineering E. coli to accumulate the TKDG and, 3) demonstrating the utility of the resulting TKDG over-producing strain by producing various TDP-deoxysugars. Specifically, biosynthetic genes responsible for the individual production of TDP-D- fucose, TDP-D-fucofuranose and TDP-D-olivose will be placed into inducible E. coli expression vectors and transformed into the engineered strain accumulating TKDG. We propose the resulting strains will be capable of producing at least 50 mg/L of TDP-D-fucose, TDP-D-fucofuranose and/or TDP-D-olivose in the Phase I study. In Phase II, we will scale-up production of TDP-deoxysugars and optimize chromatographic techniques for increased throughput. Furthermore, we will continue to utilize various TDP-deoxysugar biosynthetic genes for the production of amino sugars, branched-chain sugars and additional deoxysugars. In Phase III we will commercialize the technology developed by offering a wide variety of TDP-sugars for sale, carrying out custom synthesis of TDP-sugars, and carrying out custom glycodiversification projects. PUBLIC HEALTH RELEVANCE: This project is aimed toward developing E. coli as a whole-cell biocatalyst for the production of activated TDP-deoxysugars. These deoxysugars can be used to make derivatives of natural products with new therapeutic properties, for example, antibiotics that are effective against antibiotic-resistant bacteria.